Báo cáo hóa học: " Frequency and spectrum of mitochondrial 12S rRNA variants in 440 Han Chinese hearing impaired pediatric subjects from two otology clinics" ppt

RESEARCH Open Access
Frequency and spectrum of mitochondrial 12S
rRNA variants in 440 Han Chinese hearing
impaired pediatric subjects from two otology
clinics
Zhisen Shen
1
, Jing Zheng
2
, Bobei Chen
3
, Guanghua Peng
3,4
, Ting Zhang
2
, Shasha Gong
2
, Yi Zhu
2,5
,
Chuqin Zhang
3
, Ronghua Li
6
, Li Yang
6
, Jianjin Zhou
1
, Ting Cai
1
, Lihua Jin

1
, Jianxin Lu
2
, Min-Xin Guan
2,6,7*
Abstract
Background: Aminoglycoside ototoxicity is one of the common health problems. Mitochondrial 12S rRNA
mutations are one of the important causes of aminoglycoside ototoxicity. However, the incidences of 12S rRNA
mutations associated with aminoglycoside ototoxicity are less known.
Methods: A total of 440 Chinese pediatric hear ing-impaired subjects were recruited from two otology clinics in the
Ningbo and Wenzhou cities of Zhejiang Province, China. These subjects underwent clinical, genetic evaluation and
molecular analysis of mitochondrial 12S rRNA. Resultant mtDNA variants were evaluated by structural and
phylogenetic analysis.
Results: The study samples consisted of 227 males and 213 females. The age of all participants ranged from
1 years old to 18 years, with the median age of 9 years. Ninety-eight subjects (58 males and 40 females) had a
history of exposure to aminoglycosides, accounting for 22.3% cases of hearing loss in this cohort. Molecular
analysis of 12S rRNA gene identified 41 (39 known and 2 novel) variants. The incidences of the known deafness-
associated 1555A > G, 1494C > T and 1095T > C mutations were 7.5%, 0.45% and 0.91% in this entire hearing-
impaired subjects, respectively, and 21.4%, 2% and 2% among 98 subjects with aminoglycoside ototoxicity,
respectively. The structural and phylogenetic evaluations showed that a novel 747A > G variant and known 839A >
G, 1027A > G, 1310C > T and 1413T > C variants conferred increased sensitivity to aminoglycosides or
nonsyndromic deafness as they were absent in 449 Chinese controls and localized at highly conserved nu cleotides
of this rRNA. However, other variants were polymorphisms. Of 44 subjects carrying one of definite or putative
deafness-related 12S rRNA variants, only one subject carrying the 1413T > C variant harbored the 235DelC/
299DelAT mutations in the GJB2 gene, while none of mutations in GJB2 gene was detected in other 43 subjects.
Conclusions: Mutations in mitochondrial 12S rRNA accounted for ~30% cases of aminoglycoside-induced deafness
in this cohort. Our data strongly support the idea that the mitochondrial 12S rRNA is the hot spot for mutations
associated with aminoglycoside ototoxicity. These data have been providing valuable information and technology
to predict which individuals are at risk for ototoxicity, to improve the safety of aminoglycoside antibiotic therapy,
and eventually to decrease the incidence of deafness.

* Correspondence:
2
Attardi Institute of Mitochondrial Biomedicine and Zhejiang Provincial Key
Laboratory of Medical Genetics, School of Life Sciences, Wenzhou Medical
College, Wenzhou, Zhejiang, China
Full list of author information is available at the end of the article
Shen et al. Journal of Translational Medicine 2011, 9:4
/>© 2011 Shen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (htt p://creativecommons.org/licenses/by/2.0), which permits unrestricted use , distribution, and reproduction in
any medium, provided the original work is properly cited.
Background
Aminoglycosides, such as gentamicin a nd tobramycin,
are of great clinical importance for the treatment of bac-
terial infections. The use of these drugs can frequently
lead to toxicity, which involves the renal, auditory and
vestibular systems [1,2]. The renal impairment is usually
reversible, whereas the auditory and vestibular ototoxi-
city is usually irreversible. In familial cases of ototoxi-
city, aminoglycoside hypersensitivity is often maternally
transmitted, suggesting that mutation(s) in mitochon-
drial DNA (mtDNA) is one of molecular bases for this
susceptibility [1,2]. As mitochondrial ribosomes share
more simil arities to bacterial ribosomes than do cytoso-
lic counterparts, the human mitochondrial 12 rRNA was
proposed to be the primary targeting site for aminogly-
cosides [3,4]. The mutational analysis of mitochondrial
genome in several Chinese and Arab-Israeli families
with maternally transmitted aminoglycoside ototoxicity
or/and nonsyndromic deafness led to the landmark dis-
covery of the 12S rRNA 1555A > G mutation in 1993

[3]. Subsequently, the 1555A > G mutation has been
found to be responsible for both aminoglycoside-
induced and nonsyndromic hearing loss in many
families worldwide [4-10]. On the other hand, the 12S
rRNA 1494C > T mutation has been associated with
both aminoglycoside-induced and nonsyndromic hearing
loss only in some Chinese and Spanish families [11-13].
The 1555A > G and 1494C > T mutations are located
atthehighlyconservedA-siteof12SrRNA[4,11].The
A1555 and C1494 (equivalent to positions 1491 and 1409
of Escherichia coli 16S rRNA, respectively) are in apposi-
tion to each other but do not form a base-pair. The
1555A > G or 1494C > T mutation creates a new G-C or
A-U pair base-pair, thereby extending t he adjacent stem
by one nucleotide and making the secondary structure of
mitocho ndrial 12S rRNA mo re closely resemble the cor-
responding region of E. coli 16S rRNA and altering bind-
ing properties of aminoglycosides such as paromomycin,
neomycin, gentamicin, and kanamycin at the A-site of
12S rRNA [14]. Thus, the administration of aminoglyco-
side s can induce or worsen hearing loss in these subjects
carrying the 1555A > G or 1494C > T mutation. In the
absence of aminoglycosides, matrilineal relatives within
and among families carrying the 1555A > G or 1494C >
T mutation exhibited a considerable phenotypic variation
with respect to severity and age-of-onset and penetrance
of hearing loss [4-13]. Therefore, additional modifier fac-
tors such as aminoglycosides, nuclear and mitochondrial
genetic modifiers contributed to the phenotypic variabil-
ity of these mtDNA mutations [11,15-18].

However, the incidences of the 1555A > G and 1494C
> T mutations were only reported in the some cohorts
of hearing-impaired subjects [3,19-24]. As these
mutations are only responsible for a portion of patients
with hearing lo ss, it is anticipated that additional muta-
tions causing hearing loss can be found in the same
gene. In the present investigation, we carried out a sys-
tematic and extended mutational screening of 12S
rRNA gene in a cohort of 440 hearing-impaired Han
Chinese pediatric subjects from two otology clinics at
Ningbo and Wenzhou, Zhejiang Province, China. Muta-
tional analysis of 12S rRNA gene in these subjects iden-
tified the known 1555A > G and 1494C > T mutations
as well as 39 other variants. Those variants have been
further evaluated by phylogenetic analysis, structure-
function relation and allelic frequency of these variants
in the 449 Han Chinese controls from the same region.
To examine if the GJB2 gene contributed to a deafness
phenotype, we performed the mutational screening of
GJB2 gene in 39 subjects carrying the known deafness-
associated 12S rRNA mutations and 5 subjects carrying
one of 5 putative 12S rRNA mutations.
Methods
Subjects and audiological examinations
A total of 440 unrelated hearing-impaired Chinese sub-
jects, who were younger than 18 years old two otology
clinics from Zhejiang Province, were enrolled in this
study under an institutional review board-approved pro-
tocol of informed consent at the Cincinnati Children’s
Hospital Medical Center Institutional Review Board and

Ethics Committee of Wenzhou Medical College, China.
A comprehensive history and physical e xamination for
these participating subjects were performed to identify
any syndromic findings, the history of the use of amino-
glycosides, genetic factors related to the hearing impair-
ment. An age-appropriate audiological examination was
performed and this examination included pure-tone
audiometry (PTA) and/or auditory brainstem response
(ABR), immittance testing and Distortion product otoa-
coustic emissions (DPOAE). The PTA was calculated
from the a verage of the audiometric thresholds at 500,
1000, 2000, 4000 and 8000 Hz. The severity of hearing
impairment was classified into five grades: normal <26
Decibel (dB); mild = 26-40 dB; moderate = 41-70 dB;
severe = 71-90 dB; and profound >90 dB. The 449 con-
trol DNA used for screening for the presence o f
mtDNA variants were o btained from a panel of unaf-
fected Han Chinese subjects from the same region.
Mutational analysis of mitochondrial 12S rRNA gene
Genomic DNA was isolated from whole blood of parti-
cipants using Puregene DNA Isolation Kits (Gentra Sys-
tems, Minneapolis, Minnesota, USA). Subject’sDNA
fragments spanning the 12S rRNA gene were amplified
by PCR using oligodeoxynucleotides correspon ding to
Shen et al. Journal of Translational Medicine 2011, 9:4
/>Page 2 of 11
positions 618-635 and 1988-2007 [25]. Each fragment
was purified and subsequently analyzed by direct
sequencing in an ABI 3700 automated DNA sequencer
using the Big Dye Terminator Cycle (Applied Biosys-

tems, Foster City, California, USA) sequencing reaction
kit. The resultant sequence data were compared with
the updated consensus Cambridge sequence (GenBa nk
accession number: NC_012920) [26]. The homoplasmy
of the 1555A > G and 1494C > T mutations in these
subjects were determined as detailed previously [7,11].
The frequency of variants in the 12S rRNA gene in 449
Chinese control subjects was determined by direct
sequencing of PCR products as described above.
Mutational analysis of GJB2 gene
The DNA fragments spanning the entire coding region
of GJB2 gene were amplified by PCR using the following
oligodeoxynucleotides: forward-5’ TATGACACTCCC-
CAGCACAG3’ and reverse-5’GGGCAATGCTTAAAC-
TGGC3’. PCR amplification and subsequent sequencing
analysis were performed as detailed elsewhere [10]. The
results were compared with the wild type GJB2
sequence (Version 1, GenBank accession number:
M86849) to identify the mutations.
Structural analysis
The published secondary structures for the 12S rRNA
[27,28] were used to define the stem and loop struct ure.
The secondary structure of human mitochondrial 12S
rRNA was predicted by using the RnaViz program [29].
Phylogenetic analysis
A total of 14 primate mitochondrial 12S rRNA
sequences (Genbank), as shown in Table 1, were used in
the interspecies analysis. These include Homo sapiens,
Gorilla gorilla, Pan paniscus, Pan troglodytes, Pongo
pygmaeus, Pongo abelii, Hylobates lar, Macaca mulatta,

Macaca sylvanus, Papio hamadryas, Cebus albifrons,
Tarsius bancanus, Nycticebus coucang, and Lemur catta.
The conservation index (CI) was calculated by compar-
ing the human nucleotide variants with other 13 pri-
mates. The CI was then defined as the percentage of
species from the list of 14 different primates that have
the wild-type nucleotide at that position.
Results
Study samples
The study samples consisted of 227 males and 213
fem ales. The age of all participants ranged from 1 years
old to 18 years, with the median age of 9 years. All par-
ticipants were Han Chinese recruited from ENT clinics
at Ningbo and Wenzhou Cities of Zhejiang Province,
China. Based on a clinician review of the medical
record, 98 subjects (58 males and 40 females) had a his-
tory of exposure to aminoglycosides including gentami-
cin, streptomycin and kanamycin, accounting for 22.3%
cases of hearing loss in this cohort. These subjects, due
to infections or other illness, received a conventional
daily dosage of aminoglycosides (3 5 mg/kg/dose every
8hforgentamicinor1525mg/kg/doseevery12hfor
streptomycin, 15 mg/kg/dose every 8 h for kanamycin)
at younger than 10 years old. Hearing impairment
occurred from 3 days to three months after the adminis-
tration of drugs. Audiologi cal evaluation showed that 22
subjects had severe hearing loss and 76 individuals
exhibi ted profound hearing loss. Furthermor e, there was
the wide range of severity of hearing loss in 342 affected
subjects who did not have a history of exposure to ami-

noglycosides: 149 subjects exhibitedprofoundhearing
loss, 167 subjects had severe hearing loss and 26 indivi-
duals suffered from moderate hearing loss. The onset of
the hearing loss ranged from congenital to 10 years old.
Mutational analysis of mitochondrial 12S rRNA gene
Fragments spanning 12S rRNA gene were PCR-amplified
from genomic DNA of 440 hearing-impaired Chinese
subjects and each fragment was purified and sub-
sequently analyzed by DNA sequencing. C omparison of
the resultant sequence with the Cambridge consensus
sequence [26] identified 41 nucleotide changes in the 12S
rRNA gene as shown in Table 2. All the nucleotide
changes were verified by sequence analysis of both
strands and appeared to be homoplasmy. Of these, 2 sub-
jects with profound hearing loss carried the 1494C > T
mutation. Both subjects carrying the 1494C > T mutation
had a history of exposure to aminoglycosides. These
translate to a frequency of ~0.45% f or the 1494C > T
mutation in this Chinese pediatric deafness population.
Table 1 mtDNA sequence data of 14 primate species
Species name GenBank accession number
Homo sapiens NC_012920
Gorilla gorilla NC_001645
Pan paniscus NC_001644
Pan troglodytes NC_001643
Pongo pygmaeus NC_001646
Pongo abelii NC_002083
Hylobates lar NC_002082
Macaca mulatta NC_005943
Macaca sylvanus NC_002764

Papio hamadryas NC_001992
Cebus albifrons NC_002763
Tarsius bancanus NC_002811
Nycticebus coucang NC_002765
Lemur catta NC_004025
Shen et al. Journal of Translational Medicine 2011, 9:4
/>Page 3 of 11
Among these, 33 hearing-impaired subjects carrying
the 1555A > G mutation were composed of 21 subjects
who had a history of exposure to aminoglycosides and 12
individuals who did not receive aminoglycoside treat-
ment. These translate to a frequency of ~7.5% for t he
1555A > G mutation in this entire Chinese pediatric
deafness population, and approximately 21.4% in cases of
aminoglycoside ototoxicity in this Chinese pediatric
population. F urthermore, 4 subjects harbored the known
deafness-associated 1095T > C mutation [30,31] and 11
Table 2 Variants in the mitochondrial 12S rRNA gene in 440 hearing-impaired Han Chinese subjects
Position Replacement Conservation
index (%)
a
WC
base-pairs
b
Previously
reported
c
Number of
affected subjects
Percentage (%) Number of

controls (number/449)
Percentage (%)
663 A to G 78.6 ↓A-U Yes 15 3.40 5 1.1
681 T to C 85.7 ↓U-A Yes 5 1.13 8 1.8
709 G to A 64.3 ↓G-C Yes 90 20.41 102 22.7
723 A to G 28.6 Yes 2 0.45 2 0.4
735 A to G 78.6 Yes 2 0.45 5 1.11
747 A to G 100 ↓A-U No 1 0.23 0 0
752 C to T 100 Yes 26 6.12 17 3.8
789 T to C 85.7 Yes 1 0.23 1 0.2
813 A to G 28.6 Yes 1 0.23 0 0
827 A to G 92.9 Yes 16 3.63 12 2.7
839
d
A to G 78.6 ↓A-U Yes 1 0.23 0 0
929 A to T 42.9 ↓A-U No 1 0.23 0 0
942 A to G 64.3 Yes 1 0.23 0 0
951 G to A 92.9 ↓G-C Yes 2 0.45 2 0.4
953 T to C 57.1 Yes 1 0.23 0 0
961 insC 42.9 Yes 9 2.04 14 3.1
961 T to C 42.9 Yes 2 0.23 4 0.9
980 T to C 64.3 ↓U-A Yes 3 0.68 0 0
990 T to C 71.4 ↓U-A Yes 1 0.23 0 0
1005 T to C 35.7 Yes 21 4.76 22 4.9
1009 C to T 21.4 Yes 6 1.36 8 1.8
1027 A to G 92.9 Yes 1 0.23 0 0
1041 A to G 42.9 Yes 2 0.45 4 0.9
1048 C to T 57.1 Yes 10 2.27 11 2.4
1095 T to C 92.9 ↓U-A Yes 4 0.91 1 0.2
1107 T to C 85.7 Yes 36 8.39 25 5.6

1119 T to C 50.0 Yes 13 2.95 17 3.8
1187 T to C 57.1 Yes 1 0.23 0 0
1282 G to A 71.4 Yes 1 0.23 0 0
1310 C to T 85.7 ↓G-C Yes 1 0.23 0 0
1382 A to C 92.9 ↓A-U Yes 14 3.17 9 2.0
1391 T to C 64.3 Yes 1 0.23 1 0.2
1393 G to A 28.6 ↑A-U Yes 2 0.45 0 0
1413 T to C 78.6 ↑C-G Yes 1 0.23 0 0
1442 G to A 42.9 Yes 1 0.23 0 0
1462 G to A 50.0 Yes 1 0.23 00
1494 C to T 78.6 ↑U-A Yes 2 0.45 0 0
1503 G to A 50.0 ↑A-U Yes 1 0.23 0 0
1541 T to C 78.6 Yes 6 1.36 4 0.9
1555 A to G 85.7 ↑A-U Yes 33 7.5 0 0
1598 G to A 50 Yes 12 2.72 9 2.0
a
The conservation index (CI) was then defined as the percentage of the human nucleotide variants with other 14 primates that have the wild-type nucleotide at
that position.
b
Classic Watson-Crick (WC) base pair: created (↑) or abolished (↓).
c
See Ruiz-Pesini E, Wallace DC (2006) and ; />d
Known and putative pathogenic variants are indicated in boldface.
Shen et al. Journal of Translational Medicine 2011, 9:4
/>Page 4 of 11
subjects carried the putative deafness-associated muta-
tions at positio n of 961 (961insC and 961T > C)
[7,21,32,33], respectively.
In addition to the mutations mentioned above, there
were 34 known and 2 novel variants in the 12S rRNA

gene [34]. These variants were first evaluated by exam-
ining the allelic frequency in 449 Han Chinese control
population. Nineteen out of 41 variants were absent in
this Chinese control population. Of other 22 variants,
the frequencies of 8 variants were <1% in 449 Chinese
controls, while the allelic frequency of other 14 variants
was >1% in this control population. Furthermore, we
used the secondary structure of 12S rRNA [29,35] to
localize each variant with either a stem or a loop and to
analyze if the base changes within stems alter classic
Watson-Crick (WC) base pair [29,35]. As shown in
Figure 1, 23 variants were located at the loops, while 18
variants occurred in the stems of this rRNA. As shown
in Table 2 and Figure 1, 5 variants 1393G > A, 1413T >
C, 1494C > T, 1503G > A and 1555A > G created a
putative base-pairing(s), while 12 variants 663A > G,
681T > C , 709G > A, 747A > G, 839A > G, 929A > T,
951G > A, 980T > C, 990T > C, 1095T > C, 1310C > T
and 1382A > C abolished a putative base pairing(s).
This suggested that the nucleotide variants were more
frequent in loops than in stems. In addition, phyloge-
netic analysis was performed by comparing the
human 12S rRNA nucleotide variants with other 13
primates. As shown in Table 2, conservation index
(CI) among the variants ranged from 21.4% (1009C >
T variant) to 100% (752C > T and 747A > G variants).
Inparticular,CIof18variants including 1555A > G
and 1494C > T mutations were >78%, CI of other 13
variants was between 78% and 50% and CI for the
remaining variants was <50%. In addition to the

1555A > G and 1494C > T mutations, the novel 747A
> G variant and the known 839A > G, 1027A > G,
1310C > T and 1413T > C variants [22,34], which are
absentinthe449ChinesecontrolsandwhoseCIs
were >78%, were the putative deafness-associated var-
iants. On the other hand, other variants such as 663A
> G, 681T > C, 735A > G, 752C > T, 827A > G,
1107T > C and 1382A > C, whose CIs were >78%,
which were present in the controls, appeared to be
the polymorphisms.
Clinical characterization of 39 hearing-impaired Chinese
subjects carrying one of known or 12S rRNA mutations
Comprehensive medical evaluations of 33 probands car-
rying the 1555A > G mutation, two subjects harboring
the 1494C > T mutation and four individuals carrying
the 1095T > C mutation showed no other clinical
abnormalities, including diabetes, muscular diseases,
visual loss and neurological disorders. As shown in
Table 3, audiological assessments of 33 subjects carrying
the 1555A > G mutation showed that 15, 3 and 3 sub-
jects with the aminoglycoside treatments exhibited pro-
found, severe or moderate hearing loss, respectively.
Moreover, 12 indivi duals, who did not have a history of
exposure to aminoglycosides, exhibited a variety of
severity and age-of-onset o f hearing impairment. The
age-of-onset of hearing loss in these subjects ranged
from infant to 18 years, with an average of 6 years.
Audiometric studies showed that 3 individuals suffered
from profound hearing impairment, 5 subjects exhibited
severe hearing impairment, 2 probands had moderate

hearing impairment and 2 subjects exhibited mild hear-
ing impairment. Furthermore, two subjects carrying the
1494C > T mutation exhibited severe or profound hear-
ing loss, respectively. Among four subjects carrying the
1095T > C mutation, two subjects who was treated with
aminoglycosides had profound and severe hearing loss,
respectively, while two individuals who did not have a
history to exposure exhibited profound and mild hearing
impairment.
Clinical and genetic characterization of 5 hearing-
impaired Chinese subjects carrying one of 5 putative 12S
rRNA mutation
Comprehensive medical histories of 5 probands carrying
one of 5 putative 12S rRNA mutations and other mem-
bers in these families showed no other clinical abnormal-
ities, including diabetes, muscular diseases, visual loss
and neurological disorders. As show n in Table 3, two
subjects received a regular dose of gentamicin for various
illnesses at the age of 1 year, while other three subjects
did not have a history of exposure to aminoglycosides.
There was no evidence that these subjects had any
known cause to account for hearing loss. Audiological
examination indicated that 2 subjects suffered from
severe hearing loss and 3 subjects exhibited profound
hearing loss. Variable patterns of audiometric configura-
tions w ere detected in these subjects: 1 subject with
slope-shaped pattern and 4 individuals with flat-shaped
pattern. Besides t he proband, no one of the NS016 pedi-
gree carrying the 747A > G variant suffered from hearing
loss. The pedigree FE239 with three mat rilineal affected

relatives carrying the 1027A > G mutation showed sug-
gestively maternally transited hearing loss. Furthermore,
two matrilineal relatives of 14 members in the pedigree
NB005 carrying the 839A > G mutation, as shown in
Figure 2, suffered from hearing loss. In addition, four of
16 members in the pedigree ZX039 carryi ng the 1413T >
C variant experienced the loss of hearing.
Mutational analysis of GJB2 gene
To examine if the GJB2 gene contributed to a deafn ess
phenotype, we performed the mutational screening of
Shen et al. Journal of Translational Medicine 2011, 9:4
/>Page 5 of 11
GJB2 gene in 39 subjects carrying the known deafness-
associated 12S rRNA mutations and 5 subjects carrying
one of 5 putative 12S rRNA mutations. As shown in
Table 3, the subject ZX039-IV-1 carrying the 12S
rRNA 1413T > C mutation harbored the known
235DelC/299DelAT mutation in the GJB2 gene [36,37],
while none of other mutations in GJB2 gene was
detected in other 43 affected subjects. Indeed, the
absence of mutation in the GJB2 gene in those subjects
with hearing impairment indicated that the GJB2 gene
did not contribute to the deafness phenotype in those
subjects.
Figure 1 Structure and sequence variants of human mitochondrial 12S rRNA. The secondary structur e was predicted by using the RnaViz
program (De Rijk and De Wachter, 1997). The variants were indicated by arrows.
Shen et al. Journal of Translational Medicine 2011, 9:4
/>Page 6 of 11
Table 3 Summary of clinical and molecular data for 44 Han Chinese subjects carrying the putative 12S rRNA
mutations

12S rRNA
mutation
GJB2 gene
mutation
Subjects Gender Audiometic
configuration
Age-at-
onset
(years)
PTA
a
(dB)
right
ear
PTA
(dB)
left ear
Use
of
drugs
Level of
hearing
impairment
1555A > G polymorphism FE003-IV-1 M Slope 1 98 98 Yes Profound
1555A > G polymorphism FE007-IV-6 M Slope 2 100 100 Yes Profound
1555A > G polymorphism FE008-III-7 F Slope 10 58 78 No Severe
1555A > G polymorphism FE0128-IV-
1
F Slope 2 102 98 Yes Profound
1555A > G polymorphism FE019-IV-1 F Slope 2 67 82 Yes Severe

1555A > G polymorphism FE020-III-
15
M Slope 8 81 74 Yes Severe
1555A > G polymorphism FE036-III-1 M Slope 10 58 49 No Moderate
1555A > G polymorphism FE081-III-1 M Slope 16 50 56 Yes Moderate
1555A > G polymorphism FE122-III-2 F Slope 2 71 53 Yes Moderate
1555A > G polymorphism FE141-III-1 F Slope 2 24 30 No Mild
1555A > G polymorphism FE154-III-1 F Slope 18 61 60 No Moderate
1555A > G polymorphism FE160-III-1 F Slope 5 61 74 No Severe
1555A > G polymorphism FE163-III-3 M Flat 2 110 99 Yes Profound
1555A > G polymorphism FE300-II-12 F Slope 3 110 105 Yes Profound
1555A > G polymorphism FE304-II-2 F Slope 3 100 80 Yes Profound
1555A > G polymorphism FE317-III-
10
M Slope 4 94 93 Yes Profound
1555A > G polymorphism FE350-III-1 F Flat 1 100 100 Yes Profound
1555A > G polymorphism NB038-III-1 M Flat 1 90 87 Yes Profound
1555A > G polymorphism NB048-III-2 F Slope 1 78 81 No Severe
1555A > G polymorphism NB052-III-2 F Flat 1 120 102 No Profound
1555A > G polymorphism NB076-III-1 M Flat 1 118 118 Yes Profound
1555A > G polymorphism NB078-III-2 F Flat 6 110 117 Yes Profound
1555A > G polymorphism NB079-III-1 M Flat 1 102 102 Yes Profound
1555A > G polymorphism NB094-III-2 F Flat 1 117 117 Yes Profound
1555A > G polymorphism NB111-III-2 F Slope 3 83 86 Yes Severe
1555A > G polymorphism NB126-III-2 F Slope 2 84 92 No Profound
1555A > G polymorphism NB137-III-1 F Slope 2 111 115 No Profound
1555A > G polymorphism ZX019-II-2 F Slope 5 59 62 Yes Moderate
1555A > G polymorphism ZX022-III-3 M Flat 2 101 102 Yes Profound
1555A > G polymorphism ZX025-III-
14

M Flat 1 113 108 Yes Profound
1555A > G polymorphism ZX028-IV-1 F Slope 3 87 87 No Severe
1555A > G polymorphism ZX037-II-7 M Flat 5 30 27 No Mild
1555A > G polymorphism ZX047-III-1 M Slope 6 78 79 No Severe
1494C > T polymorphism FE247-III-1 M Flat 3 100 100 Yes Profound
1494C > T polymorphism NB133-II-1 M Slope 2 86 88 Yes Severe
1095T > C polymorphism FE312 F Slope 9 82 80 Yes Severe
1095T > C polymorphism NB021 M Slope 10 36 37 No Mild
1095T > C polymorphism NB067 M Flat 1 93 93 No Profound
1095T > C polymorphism NB100 F Flat 5 100 95 Yes Profound
747A > G polymorphism NS016-III-4 M Flat 1 100 80 Yes Profound
839A > G polymorphism NB005-III-1 F Flat 1 78 81 Yes Severe
1027A > G polymorphism FE239-II-1 M Slope 18 82 85 No Severe
1310C > T polymorphism NS071-IV-1 M Flat 1 91 92 No Profound
1413T > C 235DelC/
299DelAT
ZX039-IV-1 F Flat 1 114 111 No Profound
a
PTA: pure-tone audiometry; dB: decibel.
Shen et al. Journal of Translational Medicine 2011, 9:4
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Discussion
The cohort of Chinese pediatric hearing-impaired sub-
jects consisted of 98 subjects with aminoglycoside oto-
toxicity and 342 subjects, who did not have a history of
exposure to aminoglycosides. Of known deafness-
associated 12S rRNA mutations, the 1555A > G muta-
tion accounted for 7.5% cases of this Chinese clinical
population, while incidences of this mutation were
1.76% and 3.96% in two large cohorts of hearing

impaired pediatric Han Chinese subjects from schools of
deaf children [22,36]. In the present study, the inci-
dences of the 1555A > G mutation were 2.7% and 21.4%
cases of nonsyndromic and aminoglycoside-induced
hearing loss, respectively. In fact, the incidences of the
1555A > G mutation varied among different ethnic ori-
gins. With regard to the subjects with aminoglycoside
ototoxicity, the incidences of the 1555A > G mutation
were 33% in a small Japanese cohort [19] 13%, 10.4%
and 5% in three Chinese cohorts [3,21,22] and ~17% in
the two white cohorts from United States and Spain
[5,32,33]. However, the incidence o f 1555A > G muta-
tion in nonsyndromic hearing loss was much lower than
in those with aminoglycoside ototoxicity. In two white
cohorts with nonsyndromic hearing loss, the frequency
of the 1555A > G mutation varied from 0.6% to 2.5%
[20,24], while the incidence of the 1555A > G mutatio n
in several Asian cohorts ranged from 2.9% to 5.3%
[19,21-23]. Thus, the large proportion of subjects with
aminoglycoside ototoxicity in this cohort may contribute
to higher incidence of the 1555A > G mutation than
other cohorts. On the other hand, the incidences of the
1494C > T mutation appeared to be lower than those of
the 1555A > G mutation. In this cohort, two subjects
carrying the 1494C > T mutation had a history of exp o-
sure to aminoglycosides. This data appeared to be
higher than the previous reports that three familial cases
of 1340 sporadic Spanish hearing-impaired subjects car-
ried the 1494C > T mutation [12] and three cases of
1642 pediatric deaf children [22]. Therefore, these two

known 12S rRNA mutatio ns account for from 4% to 8%
cases among these Chinese hearing-impaired popula-
tions [10].
Of other known deafness-mutations, the frequency of
the 1095T > C mutation was 0.91% in this cohort. The
1095T > C mutation, whose CI was 92.9%, occurred in
one of 449 Chinese controls. This mutation has been
found in several genetically-unrelated families with non -
syndromic and aminoglycoside-induced hearing loss
[21,22,30,31]. This T-to-C transition disrupted an evolu-
tionarily conserved base-pair at stem loop o f the helix
25 of 12S rRNA [27]. This nucleotide is also located at
the P-site of ribosome, suggesting an important role in
the initiation of mitochondrial protein synthesis [31].
Furthermore, the frequency of mutations at position 961
including 961insC and 961T > C was 2.27% in this
pediatric population. Although mutations at this
NS016 with 747A>G variant NB005 with 839A>G variant
FE239 with 1027A>G variant NS017 with 1310C>T variant ZX039 with 1413T>C varian
t
*
Figure 2 Five Han Chinese pedigrees with aminoglycoside-induced and nonsyndromic hearing impairment. Hearing impaired individuals
are indicated by filled symbols. Arrowhead denotes probands. Asterisks denote individuals who had a history of exposure to aminoglycosides.
Shen et al. Journal of Translational Medicine 2011, 9:4
/>Page 8 of 11
position have been implicated to be associated with
hearing loss in different ethnic groups [21,22,32,33], the
lower CI (42.9%) and presence of 4% in the controls
indicated that mutations in this position were
polymorphisms.

A total of 4 1 (39 known and 2 nov el) variants in 12S
rRNA gene were identified in this cohort. Similar to
other mtDNA variations, these variants can be grouped
into three categories: neutral, adaptive and deleterious
[35]. To identify putative deleterious mutation, these
variants were further evaluated using following three cri-
teria: 1). Absent in the 449 Chinese controls; 2). CI is
>78%, proposed by Ruiz-Pesini and Wallace [35];
3). Potential structural and functional alterations [22].
Among these variants, 19 variant were absent in the 449
Han Chinese controls, while the frequency of other var-
iants ranged from 0.2% (13 variants such as 789T > C)
to 22.7% (709G > A variant) in this Chinese control
population. In particular, some of these variants occur-
ring at high frequencies of both control and patient
populations were the mitochondrial haplogroup specific
variants [36]. These included the 663A > G variant of
haplogroup A, the 827A > G and 1119T > C varian ts of
haplogroup B4, the 709G > A and 1598G > A variants
of haplogroup B5, the 1382A > C vari ant of haplogroup
D4, the 681T > C, 752C > T, 1048C > T and 1107T > C
variants of D5 haplogroup, the haplogroup F2 specific
variant 1005T > C, the 1041A > G variant of haplogroup
M9a, and the 1541T > C variant of haplogroup R5b
[38]. Apparently, these haplogroup specific variants were
adaptive or neutral but unlikely deleterious.
Phylogenetic analysis showed that CIs of 28 variants
were more than 78%. Despite their higher CI, the 14
variants such as 663A > G, 681T > C, 752C > T, 735A
> G, 827A > G, 1107T > C, 1382A > C and 1438A >

G were present in the controls. On the other hand, the
CIs for other 7 variants including 1555A > G and
1494C > T were at least 78% but these variants were
absent in 449 Chinese controls. Based on the predicted
secondary structure of mitochondrial 12S rRNA
[27,35], 23 variants were located at the loops and 18
variants occurred in the stems of this rRNA. Among
these variants, 11 variants including the 1095T > C
disrupted a WC base pairing(s) of 12S rRNA, while 5
variants including the 1555A > G and 1494C > T cre-
ated a novel WC base-pairing(s) of this rRNA [28,29].
In fact, the 1555A > G or 1494C > T muta tion made
the mitochondrial ribosome more bacteria-like
[4,11,14]. Functional cha racterization demonstrated
that the 1555A > G or 1494C > T mutation conferred
sensitivity to aminoglycosides [11,15,16,18]. Thus, indi-
viduals carrying either of mutations are predisposed to
hearing loss. Indeed, the novel 747A > G variant and
the known 839A > G, 1310C > T and 1413T > C
variants [22,34], which resided at the stems of 12S
rRNA, were fitted with three criteria for the patho-
genic mutations as described above. Furthermore, the
1027A > G variant, whose location was at a loop in
the 12S rRNA and whose CI was 92.9%, was absent in
449 Han Chinese controls. Thus, alterations of the ter-
tiary or quaternary structure of 12S rRNA by these
putative variants may leadtosignificanteffectson
function, thereby contributing to the deafness pheno-
type. Genetic and clinical evaluations of these five
hearing-impaired Chinese subjects carrying one of 5

putative 12S rRNA muta tion were performed. The
pedigree FE239 carrying the 1027A > G mutation exhib-
ited suggestively maternally transited hearing loss, while
other four pedigrees did not have a typically maternal
inheritance of hearing loss. The presence of the known
235DelC/299DelAT mutation in the GJB2 ge ne in the
subject ZX039-IV-1 carrying the 1413T > C mutation
indicated its role in the deafness phenotype. The
absence of mutation(s) in the GJB2 gene in other four
subjects suggested the involvement of other modifier
factors in the phenotypic manifestation of these putative
deafness-associated 12S rRNA variants, as in the case of
these families carryin g the 1555A > G mutation [39].
Further genetic and biochemical characterizations were
necessary for the understanding the pathophysiology of
these putative deafness-associated 12S rRNA mutations.
Moreover, approximately 70% of subjects with amino-
glycoside-indece hearing loss in this cohort did not
carry the pathogenic 12S rRNA 1555A > G and 1494C >
T mutations as well as putative deafness-associated 12S
rRNA mutations. These data implicated the involve-
ment of other nuclear genes, besides mitochondrial 12S
rRNA mutations, in development of hearing loss in
these subjects.
Conclusions
Mutations in mitochondrial 12S rRNA g ene accounted
for approximately 30% cases of aminoglycoside-induced
hearing loss in this cohort. These results strongly sup-
port the idea that the mitochondrial 12S rRNA is the
hot spot for mutations associated with aminoglycoside

ototoxicity. These data have been providing valuable
informa tion and technolog y to predict which individuals
are at risk for otot oxicity, to impro ve the safety of ami-
noglycoside antibiotic therapy, and eventually to
decrease the incidence of deafness.
Acknowledgements
This work was supported by Public Health S ervic e grants RO1DC05230 and
RO1DC07696 from the National Institute on Deafness and Other
Communication Disorders, and grants from National Basic Research
Priorities Program of China 2004CCA02200, Ministry of Public Heath of
Zhejiang Province 2006A100, Ministry of Science and Technology of
Zhejiang Province 2007G50G2090026 and Zhejiang Provincial Program for
Shen et al. Journal of Translational Medicine 2011, 9:4
/>Page 9 of 11
the Cultivation of High-l evel Innov ative Health talents to M.X.G. and
Ministry of Science and Natural Science Foundation of Zhejiang Province
Y207307 to Y.Z.
Author details
1
Department of Otolaryngology, Ningbo Medical Center, Li Huili Hospital,
Ningbo, Zhejiang, China.
2
Attardi Institute of Mitochondrial Biomedicine and
Zhejiang Provincial Key Laboratory of Medical Genetics, School of Life
Sciences, Wenzhou Medical College, Wenzhou, Zhejiang, China.
3
Department
of Otolaryngology, the Second Affiliated Hospital, Wenzhou Medical College,
Wenzhou, Zhejiang, China.
4

Department of Otolaryngology, Yuyao People’s
Hospital, Yuyao, Zhejiang, China.
5
Department of Otolaryngology, the First
Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang, China.
6
Department of Human Genetics, Cincinnati Children’s Hospital Medical
Center, Cincinnati, Ohio 45229, USA.
7
Deparment of Pediatrics, University of
Cincinnati College of Medicine, Cincinnati, Ohio, USA.
Authors’ contributions
The work presented here was carried out in collaboration between all
authors. ZS, BC, GP, YZ, CZ, JZ, TC LJ participated in the clinical data
collection. JZ, TZ, SG, RL, LY performed the mitochondrial 12S rRNA
sequence analysis and data collection. JL participated in the design of the
study. MXG conceived of the study, participated in its design and
coordination and drafted the manuscript. All authors read and approved the
final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 23 August 2010 Accepted: 4 January 2011
Published: 4 January 2011
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Cite this article as: Shen et al.: Frequency and spectrum of

mitochondrial 12S rRNA variants in 440 Han Chinese hearing impaired
pediatric subjects from two otology clinics. Journal of Translational
Medicine 2011 9:4.
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